The present invention relates to the transformation of maize genotypes by microprojectile bombardment.
The use of genetic engineering to introduce new agronomically important traits into maize such as insect resistance will have many commercial benefits. In order to accomplish this in the most expedient fashion it is necessary to have a method of transformation that can be used with maize genotypes that are commercially valuable.
The majority of instances of maize transformation have used a genotype known as A188, or derivatives of A188. This is because these lines are easily established in vitro as an embryogenic line that forms Type II, or friable, embryogenic callus and suspension cultures. Such Type II cultures have been exclusively preferred as a recipient of introduced genes in transformation methods. Unfortunately, A188 is an inferior inbred for the development of commercially important hybrids. (Hodges et al., Biotechnology, 4:219, 1986). Working with such xe2x80x9cmodelxe2x80x9d maize lines as A188 is disadvantageous in that extensive breeding is usually required in order to develop maize lines with a desirable genetic composition. What is needed is a method that can be used with commercially valuable maize lines without the need for reliance on such xe2x80x9cmodelxe2x80x9d systems based on Type II or suspension cultures.
Microprojectile bombardment has been advanced as an effective transformation technique for cells, including cells of plants. In Sanford et al., Particulate Science and Technology, 5: 27-37 (1987) it was reported that microprojectile bombardment was effective to deliver nucleic acid into the cytoplasm of plant cells of Allium cepa (onion). Christou et al., Plant Physiology 87: 671-674 (1988) reported the stable transformation of soybean callus with a kanamycin resistance gene via microprojectile bombardment. Christou et al. reported penetration at approximately 0.1 to 5% of cells. Christou further reported observable levels of NPTII enzyme activity and resistance in the transformed calli of up to 400 mg/L of kanamycin. McCabe et al., Bio/Technology 6: 923-926 (1988) report the stable transformation of Glycine max (soybean) using microprojectile bombardment. McCabe et al. further report the recovery of a transformed R1 plant from an R0 chimeric plant.
Transformation of monocots and, in particular, commercially valuable maize lines, has been problematic. Although there have been several reports of stable plant transformation utilizing the microprojectile bombardment technique, such transformation has not resulted in the production of fertile, regenerated transgenic maize plants of a commercially valuable genotype xe2x80x94each report used the genotype A188 or its derivatives (Fromm et al, BioTechnology, 8:833, 1990; Walters et al., Pl. Mol. Biol. 18:189, 1992, Gordon-Kamm et al., Plant Cell, 2:603, 1990). There are two reports of maize transformation using commercially valuable lines but both rely on the availability of Type II, friable embryogenic callus as a recipient for gene delivery (Jayne et al., 1991 Meeting of the International Society for Plant Molecular Biology, Abstract #338; Aves et al., 1992 World Congress on Cell and Tissue Culture, In Vitro 28:124A, Abstract #P-1134). Prior to the present invention, successful direct transformation of commercially valuable maize lines has not been achieved using microprojectile bombardment of immature zygotic embryos or Type I embryogenic callus.